Apparatus for Enhanced Recovery of Regenerative cells From Tissue Samples

ABSTRACT

This document describes methods and an apparatus for recovery of a cell-enriched matrix and cells (e.g., regenerative cells) from a tissue sample. In some embodiments, at least two rounds of acceleration and deceleration are performed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Nonprovisional applicationSer. No. 13/998,079, filed Sep. 27, 2013, which is a division of U.S.Nonprovisional application Ser. No. 13/329,143, filed Dec. 16, 2011, nowU.S. Pat. No. 8,951,513, issued on even date herewith, which claimspriority to U.S. Provisional Application No. 61/424,012, filed Dec. 16,2010, now expired, and to U.S. Provisional Application No. 61/555,305,filed Nov. 3, 2011, which was converted to U.S. Nonprovisionalapplication Ser. No. 13/385,599, now abandoned, and is acontinuation-in-part of both the '012 provisional application and the'599 nonprovisional application. The disclosures of the priorapplications are hereby incorporated in their entireties by reference inthe disclosure of this application. Further, this application is relatedto U.S. Nonprovisional Applications designated by Attorney Docket Nos.30254.2745DIV2 and 30254.2745DIV4, both of which were filed on even dateherewith.

TECHNICAL FIELD

This invention relates to methods and an apparatus for recovery of acell-enriched matrix and recovery of cells, and more particularly torecovery of cells from a tissue sample using two or more accelerationand deceleration steps under centrifugal force, and combinationsthereof.

BACKGROUND

Current strategies in regenerative medicine aim towards replacing tissuethat undergoes an increased apoptosis rate. That means that within theorgan there is a net loss of functional cells because more cells aredying than are being replaced. Therefore, the transfer of regenerativecells, e.g., stem cells and progenitor cells, from one location to thesite of renewal is a therapeutic approach to restore the organ back toan equilibrium. Research at our laboratories has shown that stem cellsand regenerative cells are present in every organ, primarily located inthe vascular and perivascular space with the early progenitor cells inthe vessel wall attached to the lamina elastica interna. One populationof these cells is able to replace the stroma of an organ, the other partof these cells is capable to differentiate into the respective parenchymof the specific organ. Each organ can be compared to a house, where thestroma made from fibroblasts and consisting of extracellular matrix canbe compared to walls of bricks and mortar in a house, the piping in ahouse corresponds to blood vessels of the organ and nerves represent theelectrical wiring in the walls. Inside these houses, in each organ onehas a certain type of inhabitants, such as liver cells, heart cells,bone cells, cartilage or fat cells, also called the parenchym of anorgan.

In order to restore function to a dysfunctional organ, it is importantto provide both the stroma, that means the housing that form the wallsof the organ and the inhabitants, which are the specific parenchymalcells.

A simple means to recover the regenerative cells capable of restoringorgan function is to dissociate them from subcutaneous fat tissue,because it is rich in blood vessels and the removable adipose tissue isnot essential for life. Most people are capable, even happy, to donateseveral grams of those tissues.

Autologous grafting of tissue harvested by lipoaspiration is a commonprocedure in cosmetic surgery for both small (e.g., nasolabial folds)and large (buttocks or breast) volume filling applications. The primarybenefits of this procedure termed “autologous fat grafting” are lowercost versus synthetic fillers and no immune rejection since thepatient's own tissue is used. Currently, multiple methods oflipoaspirate collection and processing are employed to obtain tissue forgrafting. Factors that determine clinical outcomes following autologousfat grafting have not been fully elucidated. However, it is widelyrecognized that improving the persistence of the graft is an area ofsignificant need.

SUMMARY

Transferred fat tissue or adipose cells recovered from one location inthe body and transferred to another continue to have an aerobicmetabolism. Without adequate blood supply these cells undergo necrosis,apoptosis, and autophagy within 24 to 48 hours and die. The problem withtraditional fat grafting, where fat is taken out by liposuction from onelocation in the body and re-injected at another location, is that agreat fraction of cells transferred do not survive and the dying cellscan cause considerable local inflammation. Persistence and regenerativepotential of the fat graft is not correlated with the content oflipid-filled adipocytes in the graft, but instead with the content ofregenerative cells such as stem cells and with the administeredextracellular matrix. Post-grafting loss of mature adipocytes is highdue to the trauma of harvest and re-administration and to the ischemicnature of the graft environment. In contrast, stem and regenerativecells are more resistant to these factors and thus contributesubstantially to long term graft viability and regenerative potential.

The present document is based on an improved method for volume fillingof subcutaneous tissue or other connective tissue structures that need avolume build up. The method includes transfer of a cell-enriched matrixthat has a reduced lipid content, but an increased concentration ofregenerative cells. It is known that connective tissue has aconsiderably long tolerance to ischemia since it has a significantlylower metabolism compared to adipocytes cells. Therefore it is the aimof the present invention to provide method and apparatus for a long-termstable volume filling with a cell-cell-enriched matrix. This results ingreater survival of the tissue when it is transplanted to a newlocation. Accordingly as described herein, neovascularization from thetissue resident stem cells provides a greater viability of thetransplanted graft. An additional improvement to this method is tore-apply a mixture of the cell-enriched matrix together with dissociatedregenerative cells recovered by the method described herein thatincludes enzymatic dissociation of lipoaspirate in a heated centrifugeby acceleration and deceleration in an inverted rotor.

Accordingly, the present document provides novel methods for preparationand recovery of a cell-enriched matrix, improved recovery ofregenerative cells from their subcutaneous location by means of the sameapparatus as used for the recovery of the matrix, and the combination ofcell-enriched matrix and regenerative cells for enhancedneovascularization and better survival of the transplanted volumefilling graft tissue.

The present document also provides provide cost effective means forrecovery of regenerative cells, which are defined as early mesenchymalcells plus the whole range of progenitor cells, from their location insubcutaneous adipose tissue. Pre-processing and reducing the content oflipid-filled cells from the initial lipoaspirate is an effective methodto save costly enzymes such as collagenase and neutral protease. Themethods include a two-step approach where the amount of lipid-filledcells in the lipoaspirate is reduced and a cell-enriched matrix isrecovered, and then subjecting the cell-enriched matrix with reducedlipid content to an enzymatic and mechanical process by using alsoincreased temperature from a heated centrifuge with a reconfigurablerotor and repeated cycles of acceleration and deceleration to recover aregenerative cellular preparation at optimized cost.

Currently known methods to process subcutaneous fat with the aim toobtain a processed lipoaspirate apply just centrifugation. As shownherein, processing by centrifugation alone can increase the cellularyield of processed tissue. However, extruding the adipose tissue beforethe centrifugation step significantly increases the cellular content ofthe processed lipoaspirate material, referred to as the cell-enrichedmatrix.

Processing of subcutaneous tissue can be performed as described hereinto yield a cell-enriched matrix, which primarily consists of collagen,laminin, elastin and other proteoglycans of the extracellular matrix andtissue resident cells, including stem and progenitor cells, collectively“regenerative cells” or “regenerative platform,” still bound in thetissue. Typically, a cell-enriched matrix contains 90% or more of theregenerative cells bound in their tissue location.

In one embodiment, the present document provides methods and anapparatus for preparing and recovering a cell-enriched matrix.

In one embodiment, the present document provides methods and anapparatus for preparing and recovering regenerative cells from thecell-enriched matrix.

In one embodiment, cellular compositions are provided that includeregenerative cells isolated as described herein, or cellularcompositions containing both a cell-enriched matrix and regenerativecells. The cell-enriched matrix prepared as described herein has areduced lipid content, but an increased concentration of regenerativecells. It is known, that connective tissue has a considerably longtolerance to ischemia since it has a significantly lower metabolismcompared to adipocytes cells. Cellular compositions described herein canenhance neovascularization of grafts and increase long term survival ofthe graft. Cellular compositions containing a combination of thecell-enriched matrix and regenerative cells are particularly useful forenhancing neovascularization of grafts and increasing long term survivalof the graft.

In one embodiment, this document features a method for recoveringcell-enriched matrix from tissue. The method includes extruding a tissuesample that contains a suspension of adipose tissue pieces in an aqueousfluid through an ostium and centrifuging the extruded tissue sample toisolate a cell-enriched matrix. The ostium can be from 1 to 5 mm indiameter. The extruded tissue sample is centrifuged for at least fiveminutes at a minimum of 400×g, preferably at higher g force up to 1200×g(e.g., 400×g to 1200×g). Cells can be recovered from the cell-enrichedmatrix as described herein.

In another aspect, this document features a method for recovering cellsfrom tissue. The method includes providing an extruded tissue samplehoused in a container adapted for a centrifuge, the tissue samplecomprising a suspension of tissue pieces in an aqueous fluid; subjectingthe sample to at least one acceleration and deceleration step usingcentrifugal force applied through a rotating element, wherein therotating element comprises a shaft and one or more arms that extend fromthe shaft, wherein (i) the one or more arms are supported from the shaftin such a manner that when the shaft rotates, the one or more arms swingupward and outward relative to the shaft or (ii) the one or more armsare supported at a fixed angle, wherein the containers attached to saidarms are held in such a position that gravitational force on material isopposite of applied centrifugal force, wherein said applied centrifugalforce ranges from about 50 g to about 4000 g. The temperature of thesample can be maintained between 32° and 42° C. One or more enzymes(e.g., proteases such as collagenases or neutral proteases, or otherenzymes as described herein) also can be included.

In one embodiment, this document features a method for recovering aregenerative platform from a tissue sample (e.g., lipoaspirate, adiposetissue, and combinations thereof). The method includes providing atissue sample housed in a first tissue collection container adapted foran automated tissue processing unit, wherein the automated tissueprocessing unit comprises a removable rotating apparatus comprising atleast two cavities, wherein each cavity is configured for detachablyinserting a tissue collection container within the cavity wherein thetissue sample comprises a suspension of tissue pieces in an aqueousfluid; and subjecting the tissue sample to at least one round ofcentrifugation of at least 400×g for at least about 5 minutes using theautomated tissue processing unit, thereby separating a cell-enrichedmatrix from the tissue sample, wherein the cell-enriched matrixcomprises a regenerative platform. The method further can includeextruding the tissue sample through an orifice prior to placing thetissue sample into the automated tissue processing unit. Thecell-enriched matrix can have a higher concentration of the regenerativeplatform compared to an otherwise corresponding method absent theextruding the tissue sample through an orifice. The method further caninclude transferring the tissue sample concentrate from the first tissuecollection container into a second collection container by a closedsystem method. In some embodiments, at least one protease can be addedto the second collection container. The cell-enriched matrix can besubjected to at least two rounds of acceleration, wherein each round ofacceleration is followed by a round of deceleration, therebydisaggregating the cell-enriched matrix. In some embodiments, thecell-enriched matrix can be filtered to obtain an injectableregenerative platform.

In any of the methods described herein, the method further can includeadministering at least a portion of the injectable regenerative platforminto a subject at an injection site, whereby the injection alters anarea at or near the injection site.

This document also features a method for disaggregating a cell-enrichedmatrix having a regenerative platform therein, wherein the methodcomprises providing a cell-enriched matrix housed in a second tissuecollection container adapted for an automated tissue processing unit,wherein the tissue collection container comprises at least one protease;and subjecting the cell-enriched matrix to at least two rounds ofacceleration, wherein each round of acceleration is followed by a roundof deceleration, and wherein at least two rounds of acceleration anddeceleration are performed at a rate of at least 10×g therebydisaggregating the cell-enriched matrix. The method further can includefiltering, or filtering and concentrating, the disaggregatedcell-enriched matrix to obtain an injectable regenerative platform.

This document also features a removable rotating apparatus comprising atleast two cavities, wherein each cavity is configured for detachablyinserting a tissue collection container within the cavity, wherein theremovable rotating apparatus is configured to rotate within an automatedtissue processing unit for separating a cell-enriched matrix from atissue sample. The removable rotating apparatus can include aradio-frequency identification (RFID) tag attached thereto that allowsthe removable rotating apparatus to be identified by the automatedtissue processing unit. Alternatively, the type of removable rotatingapparatus may be identified based on the amount of electrical currentrequired to accelerate the apparatus during the acceleration phase. Theremovable rotating apparatus can include autoclavable materials.

In another aspect, this document features an automated tissue processingunit for isolating a cell-enriched matrix from a tissue sample. Theautomated tissue processing unit can include a removable rotatingapparatus comprising at least two cavities, wherein each cavity isconfigured for detachably inserting a tissue collection container withinthe cavity. The automated tissue processing unit can include atemperature control device. The automated tissue processing unit can beconfigured to have at least two stop-start intervals of acceleration.The removable rotating apparatus can have at least one pre-determinedspecification that allows the automated tissue processing unit toidentify the removable rotating apparatus.

In another aspect, a modified centrifuge is provided that can be used toperform at least two series of rapid acceleration and deceleration stepsunder centrifugal force. Such steps can be performed in a thermallyregulated environment (e.g., 35-42° C.) in the presence of one or moreenzymes (e.g., a collagenase and a neutral protease) to enhance thedegradation of the extracellular matrix and release of cells.Centrifugation can be used to recover cells released from theextracellular matrix. The methods and apparatus described herein can beused to process any human or animal tissue that contains blood vessels.The methods and apparatus are particularly useful for recovering cellsfrom adipose tissue (e.g., subcutaneous or intra-abdominal adiposetissue), which is rich in vascularization and easy to recover from asubject.

This document also provides a method for recovering cells from tissue.The method includes providing a tissue sample housed in a containeradapted for a centrifuge, the tissue sample including a suspension oftissue pieces in an aqueous fluid; and subjecting the tissue sample to aplurality of acceleration and deceleration steps using centrifugalforce. The tissue sample can include human tissue or animal tissue, andcan contain blood vessels. The tissue sample can be adipose tissue suchas lipoaspirate. The method can include maintaining a temperature offrom 26° C. to 42° C. inside the container while subjecting the tissuesample to the plurality of acceleration and deceleration steps. Thetissue sample can be subjected to the plurality of acceleration anddeceleration steps in the presence of one or more enzymes (e.g., acollagenase, other protease, or a mixture thereof).

In some embodiments, each of the acceleration steps can be performed for5 to 20 seconds and each of the deceleration steps can be performed for3 to 20 seconds. The tissue sample can be subjected to the plurality ofacceleration and deceleration steps for 5 minutes to 180 minutes (e.g.,20 minutes to 60 minutes). In one embodiment, the tissue sample issubjected to at least three cycles of acceleration to 200×g anddeceleration to 1×g per minute for 30 minutes.

In another aspect, this document features a method for recovering cellsfrom tissue. The method includes providing a tissue sample housed in acontainer adapted for a centrifuge, the tissue sample including asuspension of tissue pieces in an aqueous fluid; subjecting the sampleto a plurality of acceleration and deceleration steps using centrifugalforce applied through a rotating element, wherein the rotating elementcomprises a shaft and one or more arms that extend from the shaft,wherein (i) the one or more arms are supported from the shaft in such amanner that when the shaft rotates, the one or more arms swing upwardand outward relative to the shaft or (ii) the one or more arms aresupported at a fixed angle, wherein the containers attached to the armsare held in such a position that gravitational force on material isopposite of applied centrifugal force, wherein the applied centrifugalforce ranges from about 50 g to about 4000 g. Each of the accelerationsteps can be performed for 5 to 20 seconds. Each of the decelerationsteps can be performed for 3 to 20 seconds. The tissue sample can besubjected to the plurality of acceleration and deceleration steps for 5minutes to 180 minutes (e.g., 20 minutes to 60 minutes). In oneembodiment, the tissue sample is subjected to at least three cycles ofacceleration to 200×g and deceleration to 1×g per minute for 30 minutes.

This document also features a method for recovering regenerative cellsfrom tissue. The method includes providing a tissue sample housed in acontainer adapted for a centrifuge, the tissue sample comprising asuspension of tissue pieces in an aqueous fluid; subjecting the sampleto a plurality of acceleration and deceleration steps using centrifugalforce; and centrifuging the sample at 400 to 4000×g to isolate cellularcomponents. The sample, when subjected to centrifugation at 400 to4000×g, can be housed within a container that includes an elongatedcylindrical central portion; a first end portion integrally formed withthe central portion; and a second open end portion integrally formedwith the central portion, wherein the first end portion narrows down toa narrow opening, and comprises a collection portion protruding from theend portion at the narrow opening, wherein the collection portion iscapable of receiving and storing a liquid and comprises a removable plugto seal the first end portion from the collection portion.

In any of the methods described herein, the container can include aporous insert, wherein the porous insert is composed of a biocompatiblematerial and having a pore size ranging from 0.5 mm to 5 mm, wherein theporous insert enhances the dissociation of cells from the extracellularmatrix of the tissue sample when the tissue sample is subjected to saidplurality of acceleration and deceleration steps. The porous insert canbe substantially cylindrical in shape, an inverted substantially conicalshape, or can bisect the container into upper and lower portions.

In any of the methods described herein, the container can include aplurality of particles, wherein the particles are at least 100micrometer in diameter and composed of one or more biocompatiblematerials, wherein the particles enhance the dissociation of cells fromthe extracellular matrix of the tissue sample when the tissue sample issubjected to the plurality of acceleration and deceleration steps. Theplurality of particles can include particles of different specificgravities or shapes.

In any of the methods described herein, the container can include ashaft disposed vertically in the internal lumen of the container, theshaft further including a plurality of arms disposed along a length ofthe shaft and extending substantially radially from the shaft into thelumen of the container, wherein the arms enhance the dissociation ofcells from the extracellular matrix of the tissue sample when the tissuesample is subjected to the plurality of acceleration and decelerationsteps. The arms can be of different shapes or sizes. The container caninclude a removable lid, wherein the shaft is affixed to the removablelid. The shaft can be rotatably affixed to the container. The shaft canbe moveable within the lumen of the container.

In another aspect, this document features a container or containerassembly that includes an elongated cylindrical central portion; a firstend portion integrally formed with the central portion; a second openend portion integrally formed with the central portion; and a portextending radially outward from the elongated cylindrical portion,wherein the first end portion narrows down to a narrow opening, andincludes a collection portion protruding from the end portion at thenarrow opening, wherein the collection portion is capable of receivingand storing a fluid and comprises a removable plug to seal the first endportion from the collection portion. The removable plug can allow fluidto flow from the end portion into the collection portion uponcentrifugal force, pressure, dissociation with an enzyme, or physicalremoval. The collection portion can be detachable from the first endportion. The collection portion can include an aqueous fluid. The secondopen end includes a mating portion.

This document also features a container assembly that includes a firstcontainer; a second container; and a coupling device adapted to couplethe first container to the second container. The first container isdescribed above. The second container includes an elongated cylindricalcentral portion, a closed end portion integrally formed with the centralportion, and an open end portion integrally formed with the centralportion, wherein the open end portion of the second container comprisesa mating portion; and the coupling device comprising a tubular centralportion with first and second open ends and a porous insert extendinghorizontally across the coupling device, wherein each open end of thecoupling device comprises a mating portion, wherein the porous inserthas a pore size of 40 to 500 μm. The coupling device further includes aport extending radially outward from the tubular central portion,wherein one mating portion of the coupling device is attached to themating portion of the first container and the other mating portion ofthe coupling device is attached to the mating portion of the secondcontainer. The port can include a porous insert.

The first and second containers can be pre-assembled, wherein aninterior space defined by the first container and the second containeris at least partially under vacuum.

This document also features a container that includes an elongatedcylindrical central portion defining an internal lumen; a first endportion integrally formed with the central portion; a second open endportion integrally formed with the central portion; a shaft disposedvertically in the internal lumen; and a plurality of arms disposed alonga length of the shaft and extending in a substantially radial directionfrom the shaft into the internal lumen. The plurality of arms can be ofdifferent shapes or sizes. The container further can include a removablelid that attaches to the second open end portion, wherein the shaft isaffixed to the removable lid. The shaft can be rotatably affixed to thecontainer. The shaft can be moveable within the lumen of the container.

In another aspect, this document features a kit that includes any of thecontainers or container assemblies described herein. The kit further oneor more cell separation reagents.

In another aspect, this document features a method to increase thecellular content in a processed lipoaspirate with the aim to recover acell enriched matrix for re-application to a patient, the methodincludes extruding the lipoaspirate through an orifice of a defineddiameter in the range of 1-5 mm, and then subjecting the extrudedlipoaspirate to a continuous centrifugation step of at least fiveminutes and a g-force of a minimum of 400×g. The centrifugation step caninclude centrifugal force of up to 2000×g. The centrifugation step caninclude centrifugal force of about 1200×g. The tissue sample can includelipoaspirate, adipose tissue, and combinations thereof. Thecentrifugation can be performed using a fixed angle, horizontal rotor.

In another aspect, this document features a method of facilitatedrecovery of regenerative cells from adipose tissue comprisingaccelerating and decelerating the tissue in the presence of proteolyticenzyme within a centrifuge, whereby the container for the tissue isinverted. The interior of the centrifuge can be temperature controlled.One or more cycles per minute of acceleration and deceleration can beapplied to the tissue. The proteolyic enzyme can be a collagenase, aneutral protease or both. A combination of collagenase and a neutralprotease can be used together with increased temperature and agitationby centrifugal acceleration and deceleration in an inverted rotor forfacilitated recovery of regenerative cells from adipose tissue.

This document also features a method of cost effective recovery ofregenerative cells from adipose tissue comprising providing a cellenriched matrix; accelerating and decelerating the cell-enriched matrixin the presence of proteolytic enzyme within a centrifuge, whereby thecontainer for the tissue is inverted. The interior of the centrifuge canbe temperature controlled. One or more cycles per minute of accelerationand deceleration can be applied to the cell-enriched matrix. Acombination of collagenase and a neutral protease can be used togetherwith increased temperature and agitation by centrifugal acceleration anddeceleration in an inverted rotor for facilitated recovery ofregenerative cells from cell-enriched matrix.

This document also features a composition containing a cell-enrichedmatrix prepared as described herein together with a regenerative cellpreparation prepared as described herein for injection into a patient.

In another aspect, this document features a removable rotating apparatuscomprising at least two cavities, wherein each cavity is configured fordetachably inserting a tissue collection container within the cavity,wherein the removable rotating apparatus is configured to rotate withinan automated tissue processing unit for separating a cell enrichedmatrix from a tissue sample. The removable rotating apparatus comprisesa radio-frequency identification (RFID) tag attached thereto that allowsthe removable rotating apparatus to be identified by the automatedtissue processing unit. The removable rotating apparatus can includeautoclavable materials.

This document also features an automated tissue processing unit forisolating a cell enriched matrix from a tissue sample, wherein theautomated tissue processing unit comprises a removable rotatingapparatus comprising at least two cavities, wherein each cavity isconfigured for detachably inserting a tissue collection container withinthe cavity. The automated tissue processing unit comprises a temperaturecontrol device. The removable rotating apparatus has at least onepre-determined specification that allows the automated tissue processingunit to identify the removable rotating apparatus.

In another aspect, this document features a method for recovering cellsfrom adipose tissue. The method including extruding lipoaspirate throughan ostium; centrifuging the extruded lipoaspirate to produce a cellenriched matrix; and subjecting the cell enriched matrix to a pluralityof acceleration and deceleration steps using centrifugal force torecover regenerative cells from the cell enriched matrix. The methodfurther can include maintaining a temperature of from 26° C. to 42° C.inside the container while subjecting the tissue sample to the pluralityof acceleration and deceleration steps. The tissue sample can besubjected to the plurality of acceleration and deceleration steps in thepresence of one or more enzymes (e.g., a collagenase, other protease, ora mixture thereof).

In another aspect, this document features a method for recovering cellsfrom tissue. The method includes providing a tissue sample housed in acontainer adapted for a centrifuge, the tissue sample comprising asuspension of tissue pieces in an aqueous fluid; subjecting the sampleto at least one acceleration and deceleration step using centrifugalforce applied through a rotating element, wherein the rotating elementcomprises a shaft and one or more arms that extend from the shaft,wherein (i) the one or more arms are supported from the shaft in such amanner that when the shaft rotates, the one or more arms swing upwardand outward relative to the shaft or (ii) the one or more arms aresupported at a fixed angle, wherein the containers attached to said armsare held in such a position that gravitational force on material isopposite of applied centrifugal force, wherein said applied centrifugalforce ranges from about 50 g to about 4000 g. The temperature of thesample can be maintained between 32° and 42° C. The tissue samplefurther can include one or more proteases.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the exemplary methods andmaterials are described below. All publications, patent applications,patents, Genbank® Accession Nos, and other references mentioned hereinare incorporated by reference in their entirety. In case of conflict,the present application, including definitions, will control. Thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a perspective view of an apparatus for dissociation,separation and recovery of cells from a tissue sample according to anembodiment described herein.

FIG. 2 is a graph of different time and energy cycles during thedissociation phase and combinations thereof.

FIGS. 3A-3E are perspective views of different containers adapted for acentrifuge.

FIG. 4 is a perspective view of a container assembly according to oneembodiment described herein.

FIG. 5 is a side cross-sectional view of one embodiment of the apparatusof FIG. 1.

FIG. 6 is a bar graph of the number of adherent cells obtained afterprocessing using an embodiment of the apparatus of FIG. 1 or afterprocessing with a shaking incubator.

FIG. 7 is a bar graph of the number of adherent cells obtained afterprocessing using an embodiment of the apparatus of FIG. 1 and containersof FIG. 3A, 3D, or 3E, or after processing using a shaking incubator.

FIG. 8 is a top view of a removable rotating apparatus that may beinserted into an automated tissue processing unit.

FIG. 9 is a side view of a removable rotating apparatus that may beinserted into an automated tissue processing unit.

FIG. 10 is a graph of the number of adherent cells/g tissue obtainedfrom adipose tissue that was treated as follows: not centrifuged,centrifuged, or extruded then centrifuged.

FIG. 11 is a graph of the number of cells/g tissue obtained from adiposetissue that was not extruded, extruded through an emulsion needle (SSextruded), or Luer extruded, and then either not centrifuged orcentrifuged at 1200×g. For extrusion the tissue was passed 5× across theextrusion device using syringes

FIG. 12 is a graph of the number of adherent cells/g tissue obtainedfrom adipose tissue after 5, 10, or 20 minutes of centrifugation.

FIG. 13 is a graph of the number of adherent cells/g tissue obtainedfrom adipose tissue after no centrifugation, centrifugation at 400×g for30 minutes, or 1200×g for 30 minutes.

FIG. 14 is a schematic of a method to increase the concentration ofadipose derived regenerative cells (ADRC) in a tissue preparation forgrafting. Tissue is first processed to concentrate ADRC in thecell-enriched matrix (CEM). One-half of the CEM is processed to yieldisolated ADRC, which are then combined with the remaining CEM to furtherincrease the concentration of ADRC in the graft.

FIG. 15 is a schematic of a method to increase the efficiency of enzymeand disposable utilization in processing of adipose tissue orlipoaspirate to obtain ADRC. Tissue is first processed to concentrateADRC in CEM, which is then combined and processed to yield ADRC.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

In general, this document is based on methods and apparatus for recoveryof cells from tissues, including human and animal tissues such ascanine, feline, equine, bovine, ovine, or porcine tissues. The methodsand apparatus described herein are particularly useful for recovery ofcells from adipose tissue obtained from, for example, liposuction (i.e.,lipoaspirate), including suction assisted, vapor assisted, or ultrasoundassisted liposuction, and combinations thereof. For instance, themethods and apparatus described herein can be used to isolate stemcells, progenitor cells, hematopoietic cells, or fully differentiatedcells from adipose tissue.

The methods and apparatus described herein can be used on site toprepare cellular compositions for administration to a patient (e.g.,autologous administration). For example, the methods and apparatusdescribed herein can be used to recover regenerative cells from apatient, referred to herein as a regenerative platform, that can beprepared for administration and then administered (e.g., injected orsurgically implanted) back to the patient from which the cells wererecovered. In some embodiments, the cells can be loaded into a deliverydevice such as a syringe, for injection into the recipient by, forexample, subcutaneous, intravenous, intramuscular, or intraperitonealtechniques. For example, the regenerative cells can be injected intoblood vessels for systemic or local delivery, into tissue (e.g., cardiacmuscle or skeletal muscle), into the dermis (subcutaneous), into tissuespace (e.g., pericardium or peritoneum), or other location. Injection ofthe regenerative platform can result in an area near the injection sitebeing augmented, repaired, having reduced inflammation, reduced pain,and combinations thereof. In some embodiments, one or more additives areadded to the cells before administration. For example, the cells can bemixed with other cells, biologically active compounds, biologicallyinert compounds, demineralized bone, a matrix or other resorbablescaffold, one or more growth factors, or other additive that can enhancethe delivery, efficacy, tolerability, or function of the cellpopulation.

The methods and apparatus also can be used to prepare cellularcompositions for growth studies, gene expression studies,differentiation studies, or other research purposes. In addition, themethods and apparatus described herein can be used to recoverregenerative cell populations (e.g., stem cells) such that the cells canbe banked, for example, by cryopreserving the cells with an appropriatemedium. For further reference, see U.S. Patent Application PublicationNo. 20100285588-A1.

In one embodiment, the methods described herein use a plurality of,i.e., two or more, acceleration and deceleration steps under centrifugalforce to enhance the dissociation of the cells (e.g., regenerative cellssuch as stem or progenitor cells) from the extracellular matrix of thetissue. One acceleration and deceleration step under centrifugal forcecan be referred to as one round of centrifugation. In some embodiments,one or more of the following also can be used to enhance recovery of thecells: mechanical disruption, mechanical agitation, maintaining thetemperature above room temperature (≧26° C.) and at or below 42° C.(e.g., about 37° C. to 40° C.), using enzymes to degrade the tissue, andseparating different components of the tissue based on physicalcharacteristics such as density, specific weight, and solubility. Insome embodiments, the tissue is mechanically disrupted (e.g., byextrusion) before subjecting the tissue to two acceleration anddeceleration steps under centrifugal force. In some embodiments, aftermechanical disruption of the tissue, a first acceleration anddeceleration step is performed under centrifugal force to separate thetissue into three general layers (i.e., an aqueous layer, acell-enriched matrix containing the regenerative platform, and a lipidlayer). The cell-enriched matrix can be removed, and subjected to asecond acceleration and deceleration step under centrifugal force. Insome embodiments, one or more enzymes (e.g., proteases) are added to thecell-enriched matrix before subjecting the cell-enriched matrix to thesecond acceleration and deceleration step. Such a process reduces thesample volume such that a smaller amount of enzymes is required toprocess the tissue. In some embodiments, a portion of the cell-enrichedmatrix is subjected to a second acceleration and deceleration step undercentrifugal force, and the regenerative cells isolated from the cellpellet. The cell isolated from the portion of the cell-enriched matrixthen can be combined with the cell-enriched matrix that has not beensubjected to further centrifugation.

Using a reconfigurable centrifuge rotor that can be configured in aninverted position or in a swinging bucket configuration as describedbelow is particularly useful in the methods described herein. When inthe inverted configuration, centrifugal forces drive contents toward theouter, higher portion of a sample container, and at rest these contentsreturn to the inner, lower portion of the sample container. Thus, byrepeated cycles of acceleration and deceleration, an inverted rotorprovides a means for agitation of a sample in a way, that at rest withgravitation and no acceleration forces, the contents of a containerreside and move towards the center and axle of a centrifuge while withacceleration, the contents follow the centrifugal forces and go outward.When tissue is combined with a proteolytic enzyme solution as describedherein, and placed in a container in the reconfigurable rotor ininverted position, repeated cycles of acceleration and decelerationfacilitate the enzymatic dissociation of the tissue. When thereconfigurable rotor is configured in a typical swinging bucket rotorconfiguration, however, the contents of the sample container concentrateat the bottom and agitation therefore is reduced.

In order to significantly improve the recovery of regenerative cells andshorten the onsite application of the methods described herein in theoperating theater, increasing the ambient temperature inside thecentrifuge chamber during repeated cycles of acceleration anddeceleration to 35 to 42° C. (e.g., 40° C.) increases the speed ofenzymatic dissociation by several fold, compared to room temperature

The methods and apparatus described herein increase the yield of cellsrecovered from the tissue sample relative to other methods in which aplurality of acceleration and deceleration steps under centrifugal forceare not utilized. The increased yield of cells recovered using themethods and apparatus described herein are surprising in view of thefindings by Kurita et al., Plast. Reconst. Surg., 121:1033-1041 (2008)in which centrifugation alone at constant speed is not sufficient torelease increased numbers of viable stem cells from the extracellularmatrix.

FIG. 1 is a perspective view of one embodiment of a centrifuge 1 havingan inner chamber separated by a wall 2 (e.g., metallic wall) therebycreating an inner container 4. Typically, the diameter of the innercontainer 4 is 20-40 cm, depending on the size of the containers usedfor the cellular preparation. Within inner container 4 is an axle 6driven by a motor 8 to rotate axle 6. The motor 8 typically is locatedbelow inner container 4. A controller 10 can turn the motor 8 on or off,and also can serve to regulate temperature inside inner container 4 asdescribed below. Motor 8 through axle 6 turns rotor 12, which has two(as depicted) or more arms, each of which is capable of receiving andgripping container 16 in a firm link to the rotor arm 14. In oneembodiment, rotor arm 14 can swing up and down based on the centrifugalforces exerted through motor 8 via axle 6, and depending on their speed,change their position from a vertical position to a fully 90 degreeposition when a certain g force (e.g., 20-30 g) is exceeded by therotations per minute of the axle 6. In another embodiment, such as thatdepicted in FIG. 5, rotor arm 14 is set at a fixed angle θ (e.g., anangle ranging from 1 degree to less than 90 degrees). For example, theangle θ can be fixed at 12 degrees. In such an embodiment, container 16can be attached to rotor arm 14 in an inverted orientation in which thetop of container 16 containing a removable lid is oriented near thecenter by axle 6.

In some embodiments, the temperature of inner container 4 can beregulated such that the temperature of the inner container ranges, forexample, from 26 to 42° C., 30 to 42° C., 35 to 42° C., 35 to 40° C., 37to 40° C. or about 37° C. The temperature can be regulated by any knownmethod, e.g., closed loop thermal feedback regulation. In oneembodiment, electrical resistance wires (not shown in FIG. 1) can bewrapped around or embedded in the inner container 4 in order to warm upthe inner container 4 through a connection to electricity, for example,via a cable. Such wires can be part of a heat pad or embedded in aflexible polymer. In order to keep the temperature constant, atemperature probe 18, which is operably linked to controller 10, can beused to sense the temperature in the inner container 4 and viacontroller 10, regulate the temperature within the inner container 4.Maintaining the temperature at 35 to 42° C. is particularly useful whenone or more enzymes are used, as temperatures below this range can slowthe dissociation process and temperatures above 42° C. can damage cells.

Controller 10 can be programmed to, for example, control theacceleration and deceleration steps, start and stop the motor 8, andregulate temperature. Controller 10 connects to a power source (e.g.,through a plug or cable).

Controller 10 can be programmed to accelerate container 16 to achieve ag force of between 50×g and 4,000×g inclusive, maintain that g force fora short period of time, and decelerate the container 16 to 1×g within ashort period of time. Repetitive cycles of the acceleration/decelerationsteps can be applied over a time frame from 5 to 180 minutes (e.g., 5 to120 minutes, 10 to 100 minutes, 20 to 60 minutes, 25 to 50 minutes, 30to 45 minutes, about 30 minutes, or about 45 minutes). For example, eachacceleration step can be performed for 5 to 20 seconds and eachdeceleration step can be performed for 3 to 20 seconds. In oneembodiment, at least three cycles of acceleration to 200×g anddeceleration to 1×g per minute can be performed for 30 minutes.

FIG. 2 depicts examples of various time cycles that can be used toenhance the dissociation of cells from the extracellular matrix in thetissue sample contained in container 8 as shown in FIG. 1. Differentpatterns as depicted in FIG. 2 can be combined such as intermittent onand off and certain accelerations in which a certain g force ismaintained over a longer period of time.

FIGS. 3A-3E depict containers, each of which is an exemplary embodimentof the container 16 shown in FIGS. 1 and 6. The containers in FIGS.3A-3E are adapted for use in a centrifuge (e.g., a centrifuge depictedin FIG. 1 or FIG. 6). The containers have an insert that can aid in thedissociation of the cells from the extracellular matrix of the tissuewhen the containers are subjected to a plurality of acceleration anddeceleration steps under centrifugal force, such as the centrifugalforces imparted by centrifuge 1 of FIG. 1. In each of FIGS. 3A-3E,container 300 includes an elongated cylindrical central portion 302, aclosed end portion 304 integrally formed with the central portion, andan open end portion 306 integrally formed with the central portiondefining an interior lumen 308. Closed end portion 304 can besubstantially flat, rounded, hemispherical, conical, or any otherappropriate shape. In some embodiments, the container 300 can have alength of approximately 10-12 cm. In some embodiments, the container 300can have a volume of approximately 50-60 ml.

The open end portion 306 includes a mating portion 310 (e.g., a threadedportion) that is formed to accept a removable cap 312. The removable cap312, when attached onto the mating portion 310, substantially enclosesand seals the interior lumen 308 of the container 300. In someimplementations, the cap 312 may be attached to the open end by threads,friction (e.g., a snap-on cap), by clamping, by magnetic attraction, bya vacuum seal, or by any other appropriate mechanism by which a vesselcan be reversibly sealed.

In FIG. 3A, container 300 includes an inverted substantially conicalinsert 320. The conical insert 320 is formed as an inverted hollow conewithin the interior lumen 308. The inverted substantially conical insert320 is substantially coaxial with the elongated cylindrical centralportion 302, with a conical sidewall 322 that extends from a vertex 324proximal to the enclosed end portion 304 to a base 326 proximal the openend portion 306.

The inverted substantially conical insert 320 is composed of abiocompatible material and is porous. Non-limiting examples ofbiocompatible materials include polyamides (e.g., Nylon); polyesterssuch as polycaprolactone; polystyrene; polypropylene; polyacrylates;polyvinyl compounds; polycarbonate; polyketones such aspolyetheretherketone (PEEK); polytetrafluoroethylene (PTFE, Teflon);thermanox; nitrocellulose; poly(ortho esters); polyurethane; stainlesssteel; titanium; or titania (titanium dioxide). The pore size of theinsert can range from 0.5 mm to 5 mm (e.g., 0.7 to 1.5 mm, 0.7 to 1.2mm, 0.9 to 1.1 mm, 0.9 to 1.5 mm, 0.9 mm to 2.0 mm, 1 to 3 mm, 2 to 4mm, 3 to 5 mm). In some embodiments, the inverted substantially conicalsidewall 322 can be a screen, lattice, mesh, net, perforated sheet, orother suitable biocompatible porous substrate. In some embodiments, theinverted substantially conical insert can be a mesh with 1 mm pores.

The base 326 of the conical insert 320 has a diameter that issubstantially the same as the diameter of the elongated cylindricalportion 302 at the open end 306. As such, the conical insert 320 isinserted into the interior volume 308 through the open end 306 until thebase contacts the rim of the open end 306. The cap 312 is then removablyaffixed onto the open end 306, thereby substantially centering andaffixing the conical insert 320 within the interior lumen 308. In someembodiments, the base 326 contains a flange that can be used to attachto the open end 308. Base 326 also can be secured directly to the bottomsurface of the cap 312. Base 326 also can be secured directly to end304.

In use, the inverted substantially conical insert 320 can be insertedinto the container 300. The inverted substantially conical insert 320can be filled with a tissue sample that includes a suspension of tissuepieces in an aqueous fluid, such that fluids and components of thetissue smaller than the pores are able to pass through the conicalinsert 320 to be captured by the cylindrical sidewall 304 and closed end306.

In some embodiments, one or more proteases (e.g., one or morecollagenases such as type I and/or type II collagenases, a neutralprotease such as thermolysin, trypsin, or mixtures thereof) can be addedto a container 300 to enhance the dissociation of the cells from theextracellular matrix of the tissue sample. For example, a type Icollagenase, a type II collagenase, and a dispase can be used to enhancethe dissociation of the cells from the extracellular matrix.

The cap 312, once applied, seals the interior lumen 308 andsubstantially affixes the conical insert 320 in position. The fluids andtissues are urged through the pores of the substantially conical insert320 by the plurality of acceleration and deceleration steps. Forexample, the container 300, with a tissue sample loaded within thesubstantially conical insert 320, can be attached onto the rotor arm 14of the centrifuge 1 of FIG. 1. The container 300 can then be acceleratedand decelerated as discussed in the description of FIGS. 1 and 2. Underthe repeated cycles of acceleration and deceleration, the tissue sampleis urged in various directions through the pores of the insert. Thisprocess mechanically disrupts the tissue to enhance the release of thecells from the extracellular matrix.

In FIG. 3B, container 300 include a substantially cylindrical insert340. The substantially cylindrical insert 340 is formed as a cylinderwithin the interior lumen 308. The cylindrical insert 340 issubstantially coaxial with the elongated cylindrical central portion302, with a sidewall 344 that extends from closed end portion 304 toopen end portion 306. The substantially cylindrical insert 340 iscomposed of a biocompatible material and is porous. Examples of suitablebiocompatible materials and pore sizes are discussed above.

In use, the substantially cylindrical insert 340 can be inserted intothe container 300. The substantially cylindrical insert 340 can befilled with a tissue sample containing a suspension of tissue pieces inan aqueous fluid such that fluids and components of the tissue smallerthan the pores are able to pass through the cylindrical insert 340 to becaptured by the elongated cylindrical central portion 302 and closed end304. One or more enzymes also can be added to the container as discussedabove. The cap 312, once applied, seals the interior volume 308 andsubstantially affixes the cylindrical insert 340 in position. Thecontainer 300 can then be accelerated and decelerated as discussed inthe description of FIGS. 1 and 2. Under the repeated cycles ofacceleration and deceleration, the tissue sample is urged in variousdirections through the pores of the insert. This process mechanicallydisrupts the tissue to enhance the release of the cells from theextracellular matrix.

In FIG. 3E, the container 300 contains an insert 345 that bisects thecontainer into upper and lower portions. The insert is composed of abiocompatible material and is porous. Examples of suitable biocompatiblematerials and pore sizes are discussed above. The insert can be held inplace using, for example, a ring made out of rubber. In use, the tissuesample containing the suspension of tissue pieces in an aqueous fluid isloaded into the upper or lower portion of the container and subjected tothe plurality of acceleration and deceleration steps to enhance thedissociation of the cells from the extracellular matrix. One or moreenzymes also can be added with the tissue sample.

Referring now to FIG. 3C, a container 300 includes a plurality ofparticles 350 within the interior lumen 308. The pellets are at least100 micrometers in diameter and are composed of one or morebiocompatible materials or coated with one or more biocompatiblematerials. Non-limiting examples of biocompatible materials includepolyamides (e.g., Nylon); polyesters such as polycaprolactone;polystyrene; polypropylene; polyacrylates; polyvinyl compounds;polycarbonate; polyketones such as PEEK; PTFE; thermanox;nitrocellulose; poly(ortho esters); polyurethane; stainless steel;titanium; titania (titanium dioxide); and glass. In some embodiments,the plurality of particles 350 can include particles of differentspecific gravities, shapes, or surface characteristics. In oneembodiment, the plurality of particles 350 can include smoothpolystyrene beads and particles with an iron coating.

In use, a tissue sample containing a suspension of tissue pieces in anaqueous fluid and the particles 350 are loaded into the container 300and sealed with the cap 312. In some embodiments, one or more enzymesalso are added to the container before sealing with the cap. Thecontainer is then loaded into the centrifuge 1 of FIG. 1 and issubjected to the plurality of acceleration and deceleration steps, whichcause the particles 350 to be agitated and enhance the release of thecells from the extracellular matrix.

FIG. 3D shows another example of a container 300 containing an insert inwhich a shaft 370 is disposed vertically in the internal lumen 308 ofthe container. The shaft 370 includes a plurality of arms 372 disposedalong a length of the shaft 370 and extending substantially radiallyfrom shaft 370 into the lumen 308. The arms 372 can be of differentshapes and/or sizes as depicted in FIG. 3D.

In some embodiments, shaft 370 can be affixed to removable lid 312.Shaft 370 can be rotatably affixed to the container or removablecontainer lid 312. For example, the shaft 370 can be rotatably affixedto, and extend through, the cap 312, such that the shaft 370 can begripped and rotated from outside the container to agitate the tissuesample. In some embodiments, the shaft 370 can be rotatably affixed tothe cap 312, and can be eccentrically weighted such that the shaft 370can rotate under the force of gravity or centrifugation. In someembodiments, the shaft 370 can be rotatably affixed to the cap 312, andone or more of the arms 372 can include a magnet such that the shaft 370can be magnetically coupled to a magnetic field external to thecontainer. By rotating the magnetic field relative to the container, theshaft 370 can be urged to rotate within the interior lumen 308 toagitate the tissue sample. In some embodiments, shaft 370 can bemoveable vertically within lumen 308 such that the arms 372 can pass upand down through the tissue sample. In some embodiments, the arms aresharpened blades.

In one embodiment, a tissue sample containing a suspension of tissuepieces in an aqueous fluid can be loaded into the container 300 and thecap 312 with the shaft 370 attached is affixed to seal the open end suchthat the arms 372 disposed along a length of shaft 370 are inserted intothe interior lumen 308 and the tissue sample. One or more enzymes alsocan be added with the tissue sample. The container 300 can then besubjected to a plurality of acceleration and deceleration steps asdiscussed herein. The shaft 370 and associated arms 372 can enhance thedissociation of cells from the extracellular matrix of the tissue samplewhen subjected to the plurality of acceleration and deceleration steps.

FIG. 4 shows an example of a container assembly 400 of a first container300, second container 404, and a coupling device 406. In the embodimentdepicted in FIG. 4, the assembly 400 includes the container 300 of FIG.3A and a container 404. In some embodiments, the container 300 can beany of the containers depicted in FIGS. 3B-3E. The container 404includes an elongated cylindrical central portion 408; a first endportion 410 integrally formed with central portion 408; and a secondopen end portion 412 integrally formed with central portion 408,defining an internal lumen 414.

In some embodiments, a port 432 can extend radially outward from theelongated cylindrical portion 408 and provide a fluidic passage thatextends from the interior lumen 414 to the outside. Such a port also caninclude a porous insert 434. In some embodiments, the porous insert canhave pores of approximately 0.2 microns, which can allow air to pass butprevent contaminants from entering the interior lumen 414.

The first end portion 410 narrows down to a narrow opening 416, andcontains a collection portion 418 protruding from end portion 410 at thenarrow opening 416. The collection portion 418 is capable of receivingand storing a fluid and includes a removable plug 420 to seal the firstend portion 410 from the collection portion 418. The removable plug 420allows fluid to flow from the end portion 410 into the collectionportion 418 upon centrifugal force, pressure, dissociation with anenzyme, or physical removal. In some embodiments, the removable plug isa valve that can be activated to provide access to the collectionportion. In some embodiments, the collection portion is detachable fromthe first end portion.

In some embodiments, the collection portion 418 comprises an aqueousfluid that is separated from the interior lumen 414 via removable plug420. For example, collection portion 418 can include sterile saline,buffer, cell culture medium, one or more biologically active compounds,one or more biologically inert compounds, demineralized bone, a matrixor other resorbable scaffold, one or more growth factors, or otheradditive that can enhance the delivery, efficacy, tolerability, orfunction of the cell population.

The second end portion can contain a mating portion (e.g., a threadedportion) such that a cap can be attached to substantially seal thecontainer.

Container 404 can be removably connected to container 300 using couplingdevice 406, which includes a tubular central portion 422 with first andsecond open ends 424 and a porous insert 426 extending horizontallyacross coupling device 406. Each open end 424 includes a mating portion(e.g., a threaded portion). The porous insert has a pore size of 40 to500 micrometers and extends horizontally across coupling device 406 suchthat porous insert 426 substantially separates the interior volume 308from the interior volume 414. In some embodiments, two or more porousinserts can be disposed on top of one another. For example, a porousinsert with relatively larger pore sizes can be disposed more closely tothe container 300, while the porous inserts with relatively smaller poresizes can be disposed more closely to the container 404. As such, fluidsand particles flowing from the container 300 to the container 404 canpass through progressively smaller pores as they pass through the porousinsert 426.

Coupling device 406 further can include a port 428 extending radiallyoutward from the tubular central portion 422 to provide a fluidicpassage from the interior of the coupling device. The port can include aporous insert 430 with a pore size of 0.2 to 500 micrometers. In someembodiments, porous insert 430 can have pores of approximately 0.2microns, which can allow air to enter into the interior of the couplingdevice 406, but filter out bacteria and particulate matter than couldcontaminate the containers 300, 404 or the tissue sample.

One mating portion of the coupling device can be attached to the matingportion of container 300 and the other mating portion of the couplingdevice can be attached to the mating portion of container 404. Inembodiments in which the open end portions of containers 300 and 404 arethreaded, coupling device 406 is threaded on each end to allow thecontainers 300 and 404 to be threaded into coupling device 406 by theirthreaded portions. In some embodiments, containers 300 and 404 arepre-assembled such that an interior space defined by the first containerand the second container is at least partially under vacuum.

In use, the container 300 can be uncoupled from coupling device 406 andloaded with a tissue sample, buffer (e.g., lactated Ringer's), andoptional enzyme. A cap (e.g., the cap 312) is applied to removably sealthe container 300. The container 300 and the tissue sample within can beprocessed in the centrifuge 1 as discussed above. After subjecting thesample contained within container 300 to the plurality of accelerationand deceleration steps as discussed above, the cap 312 can be removedand coupling device 406 can be attached to container 300 and container404. The liquid components within container 300 can be forced intocontainer 404 by inverting the container assembly and applying negativepressure to port 432. For example, a small piece of tubing can beattached to port 432 and suction applied using a syringe (e.g., with aLuer connection) to create a negative pressure in container 404 suchthat fluid and cells within the fluid in container 300 are forcedthrough the porous insert 426 and into the interior lumen 414 ofcontainer 404.

After transfer to container 404, container 300 can be detached from thecoupling device and the coupling device can be detached from container404. A cap can be removably attached to the mating portion 424 tosubstantially seal the container 404. Cells dissociated from theextracellular matrix then can be recovered from other cellularcomponents by centrifuging the container at 400 to 4000×g. In someembodiments, the cells are collected in the collection portion 418 ofcontainer 404.

In some embodiments, the centrifugation at 400 to 4,000×g can beperformed as a second program carried out using centrifuge 1. Forexample, the second program can be programmed into controller 10. Thecontainer 404 can be inserted into the rotor arm 14 in a fixed angleembodiment in which the cap is oriented toward the center of the rotorand the collection portion 418 is oriented away. Container 404 can becentrifuged for about 5 to 10 minutes with a g force of about 400×g to4,000×g (e.g., 400 to 1,000×g). Centrifugation at such g forces allowsfor separation and collection of cells at the collection portion 418.

In some embodiments, one or more cell separation reagents, includingmagnetic beads or antibodies or antigen binding fragments thereof can beused in conjunction with the methods and apparatus described herein. Forexample, antibodies having binding affinity for a particular cell typecan be used to recover cells of that type from cells collected withinthe collection portion. In some embodiments, such cell separationreagents are included within container 300. In some embodiments, suchcell separation reagents are included within container 404.

In some embodiments, a tissue sample is subjected to one accelerationand deceleration step under centrifugal force to prepare a cell-enrichedmatrix, which is then subjected to one or more acceleration anddeceleration steps. For example, a tissue sample housed in a tissuecollection container can be centrifuged to produce a cell-enrichedmatrix that includes a regenerative platform therein. Regenerativeplatform refers to regenerative cells such as stem cells, progenitorcells, and/or hematopoietic cells within the concentrate. An example ofsuch a cell preparation is given in US 2010/0124563 A1. The tissuesample may have a regenerative platform throughout the tissue sample;however, centrifugation causes the tissue sample to form a cell-enrichedmatrix having a concentrated amount of the regenerative platformtherein. For example, upon centrifugation of a tissue sample within atissue collection container, three general layers form (e.g., an aqueouslayer, a cell-enriched matrix having the regenerative platform, and alipid layer). The cell-enriched matrix is generally located between thelipid layer and the aqueous layer. After the extraction of the aqueouslayer, the cell-enriched matrix can be easily extracted from the tissuecollection container (e.g., using a closed system) for further use ofthe cell-enriched matrix.

An automated tissue processing unit can be used to centrifuge the tissuesample. For example, an automated tissue processing unit having aremovable rotating apparatus therein, where the removable rotatingapparatus is configured to rotate within the automated tissue processingunit, can be used to generate the centrifugal force. The automatedtissue processing unit may include a temperature control device forcontrolling the temperature within the unit.

FIG. 8 is a top view of a removable rotating apparatus 500 that may beinserted into an automated tissue processing unit (not shown). A tissuecollection container 502 may be detachably inserted into the removablerotating apparatus 500 and held in place by a locking mechanism 504(e.g., snappable locking mechanism). Here, the tissue collectioncontainer 502 snaps into the cavity 506. In one non-limiting embodiment,the tissue collection container 502 is customizable to snappably fitwithin the cavity 506 and held in place by the snappable lockingmechanism 504. The tissue collection container 502 is oriented so thatthe opening 512 of the tissue collection container 502 is farthest fromthe center. The formation of a cell-enriched matrix may form near theopening 512, and the cell-enriched matrix may be easily extracted fromthe tissue collection container 502 through the opening withoutadditional contamination to the cell-enriched matrix.

The removable rotating apparatus 500 may have at least two cavities(shown here as 506, 508) or may have up to about eight cavities inanother non-limiting embodiment. Each cavity 506, 508 may be in ahorizontal orientation and may have a detachable mechanism for insertinga tissue collection container 502 within the cavity 506, 508. Thedetachable mechanism may be, but is not limited to a snapping mechanism,Velcro, and the like, and combinations thereof. In another non-limitingembodiment, the removable rotating apparatus 500 may have or includeautoclavable materials, such that the removable rotating apparatus 500is configured to be autoclavable.

FIG. 9 is a side view of a removable rotating apparatus 500 that may beinserted into an automated tissue processing unit (not shown). Thetissue collection container 502 is shown within the cavity 506 and heldin place by the locking mechanism 504. It will be appreciated that theremovable rotating apparatus illustrated in FIGS. 8-9 is not to scale orproportion and that certain features of it may be exaggerated ordistorted for illustrative purposes.

The automated tissue processing unit may also have a mechanism foridentifying a particular removable rotating apparatus by at least onepre-determined specification of the removable rotating apparatus. In oneembodiment, the removable rotating apparatus may have an attached RFIDtag. The RFID tag may be scanned upon placement of the removablerotating apparatus into the automated tissue processing unit foridentification by the automated tissue processing unit. In anotherembodiment, a specification of the removable rotating apparatus withinthe automated tissue processing unit may be measured and recorded, suchas, but not limited to, the power demand associated with acceleration,weight, wind resistance, and combinations thereof. The measuredspecification may be stored as part of a software program and/orsoftware package of the automated tissue processing unit that enablesthe automated tissue processing unit to identify the removable rotatingapparatus by such specification data.

The tissue sample may be housed in a first tissue collection containeradapted for the automated tissue processing unit. The tissue sample mayhave or include a suspension of tissue pieces in an aqueous fluid. Inone non-limiting embodiment, the tissue sample may be extruded prior toplacement of the tissue sample into the first tissue collectioncontainer. The tissue sample may be extruded between one and twentytimes, or alternatively from about two times to about ten times throughan orifice ranging in diameter from about 1 mm independently to about 4mm, or alternatively from about 1.5 mm independently to about 3 mm.Extruding the tissue sample before subjecting it to a round ofacceleration produces a cell-enriched matrix that has a higherconcentration of the regenerative platform compared to an otherwiseidentical method absent the extrusion of the tissue sample. As usedherein with respect to a range, “independently” means that any lowerthreshold may be used together with any upper threshold to give asuitable alternative range.

The tissue sample may be subjected to centrifugation to achieve a gforce ranging from about 200×g independently to about 2000×g, oralternatively at least 400×g using the automated tissue processing unit.The centrifugation may occur for a time period ranging from about 3minutes independently to about 60 minutes, or at least about 5 minutes.After the centrifugation, a cell-enriched matrix may form.

The cell-enriched matrix can be transferred from the first tissuecollection container into a second collection container by a closedsystem method. Such a closed system method may include, but is notlimited to, a mechanism such as a leur connector between the firstcollection container and the second collection container, a spike port,a needle, or combinations thereof. The closed system method of transferprevents the cell-enriched matrix that includes a regenerative platformfrom being contaminated by any additional pathogens external to thetissue sample and/or the tissue collection containers, such as but notlimited to bacteria, viruses, and the like from entering into the tissuecollection containers or the cell-enriched matrix. The closed systemdecreases the necessity for additional steps to be performed on thecell-enriched matrix prior to the administration of the tissue sampleback into a subject as described above. As used herein, the numeralnotation of ‘first tissue sample container’ and ‘second tissue samplecontainer’ denotes the usage order of the containers. The containers maybe the same types of containers or different types of containers, e.g.,a vial or centrifuge tube.

The second tissue collection container may then be subjected to at leastone more acceleration and deceleration steps, as described above. Eachround of acceleration and deceleration may occur until at a rate of atleast about 10×g is obtained. Alternatively, the rate of accelerationand deceleration may occur at a rate ranging from about 10×g toindependently about 400×g, or from about 20×g independently to about40×g in another non-limiting embodiment. In one non-limiting embodiment,the number of rounds per minute of acceleration and deceleration mayrange from about 1 round per minute to about one round per five minutes,or alternatively at least about three rounds per minute. In anothernon-limiting embodiment, the cell-enriched matrix may be disaggregatedafter a number of rounds of acceleration and deceleration, oralternatively at least two rounds of acceleration and deceleration.

In some embodiments, one or more proteases (e.g., one or morecollagenases such as type I and/or type II collagenases, a neutralprotease such as thermolysin, trypsin, or mixtures thereof) can be addedto the second tissue container. One or more of the proteases may berecombinantly produced. For example, one or more collagenases can beadded to the second tissue collection container in an amount rangingfrom about 0.5 Wunsch units collagenase per ml independently to about4.0 Wunsch units collagenase per ml, or alternatively from about 1.0Wunsch units collagenase per ml independently to about 3.0 Wunsch unitscollagenase per ml. The intermittent rounds of acceleration followed bydeceleration in the presence of a protease may disaggregate theregenerative platform of the cell-enriched matrix.

After the acceleration and deceleration steps, the cell-enriched matrixmay be filtered and washed to obtain a regenerative platform that may beadministered back into a subject by implantation or injection asdescribed above. For example, the cell-enriched matrix can be filteredto obtain an injectable regenerative platform, in which a few or noadditional steps must be performed for the regenerative platform to beinjected into a subject.

The invention will be further described with respect to the followingExample which is not meant to limit the invention, but rather to furtherillustrate the various embodiments.

EXAMPLES Example 1

Fresh canine omental adipose tissue was obtained from tissue discardedafter spay surgery. Tissue was minced with sterile scissors and thenequally divided (approximately 2 g/tube) into 50 mL sterile centrifugetubes. Sterile lactated Ringer's containing a blend of bacterialcollagenases I and II together with dispase was added and the tubes werethen randomly assigned to incubation in a shaking incubator (60 rpm) ora heated tissue processing apparatus in a fixed rotor (TPA, 3 cycles permin of 1×g to 200×g to 1×g). Temperature was maintained between 37-40°C. and incubation/processing was conducted for 30 min.

After processing, the dissociated tissue slurry was passed through a 100μm filter and the cell fraction was recovered from the filtrate bycentrifugation at 400×g for 10 min in the TPA. Cell fractions wereplated in 25 cm² tissue culture flasks and grown for two days at 37° C.in DMEM/20% (v/v) fetal bovine serum (FBS) containing antibiotic andantimycotic. After culturing for two days, adherent cells were countedusing a hemacytometer. FIG. 6 is a bar graph of the number of adherentcells obtained after processing using the TPA and using the shakingincubator. Processing the tissue with the TPA resulted in a 2.6 foldhigher than when dissociatiion was performed in a shaking incubator.

Example 2

Fresh human adipose lipoaspirate was obtained with patient informedconsent from a patient undergoing elective lipoplasty. Tissue wasdrained using a sterile stainless steel strainer and then equallydivided (approximately 10 g/tube) into 50 ml sterile centrifuge tubeswith (see, FIGS. 3A, 3D, and 3E) or without inserts fabricated fromnylon mesh with 1 mm pore size. Sterile lactated Ringer's containing ablend of bacterial collagenases I and II together with dispase was addedand the tubes then were randomly assigned to incubation in a shakingincubator (60 rpm) or a heated TPA in a swinging bucket rotor (3 cyclesper min of 1×g to 200×g to 1×g). Temperature was maintained between37-40° C. and incubation/processing was conducted for 30 min.

After processing, the dissociated tissue slurry was passed through a 100μm filter and the cell fraction was recovered from the filtrate bycentrifugation at 400×g for 10 min in the TPA. Cell fractions wereplated in 25 cm² tissue culture flasks and grown for two days inDMEM/20% (v/v) FBS containing antibiotic and antimycotic. Afterculturing for two days, adherent cells were counted using ahemacytometer. FIG. 7 is a bar graph of the number of adherent cellsobtained after processing using the TPA and three different inserts, orafter processing using the shaking incubator. Results indicate that cellyield obtained by processing in the TPA and using the cone insert (e.g.,FIG. 3A) is similar to cell yield obtained by processing in theincubator.

Example 3

A lipoaspirate sample was obtained from a human patient undergoingelective lipoplasty. Lipoaspirate was transferred to a plurality oftissue collection containers having a 20 cc volume. Lipoaspiratecontents of each of the tissue collection containers were extruded fivetimes through a micro-emulsifying needle. The extruded lipoaspirate fromeach of the tissue collection containers was then transferred to aseparate tissue collection container. The tissue collection containerswere then placed into the cavities of a removable rotating apparatuswithin an automated tissue processing unit. The removable rotatingapparatus allowed the tissue collection containers to maintain ahorizontal position, while the automated tissue processing unit appliedacceleration and centrifugal force to the contents of the tissuecollection containers. The centrifugal force was applied for 30 minutesat a rate of about 400×g, about 700×g, about 1200×g, or about 2000×g for30 minutes. The contents of two tissue collection containers were usedfor each rate of centrifugal force.

After the centrifugation, a cell-enriched matrix having a regenerativeplatform was separated from the remainder of the tissue sample withineach tissue sample collection container. The volume of the layer of oil,a lipoaspirate fraction, and an aqueous fraction was removed andmeasured for each sample. The lipoaspirate layer was placed into aseparate 50 cc conical centrifuge tube.

As a control sample, approximately 5 g of unprocessed, i.e., neitherextruded nor centrifuged, lipoaspirate was transferred to each of 2tissue collection containers. The containers were weighed and the weightof the transferred lipoaspirate was recorded. Ringer's lactate thatincludes a collagenase enzyme and a dispase enzyme was added to theunprocessed lipoaspirate in an amount of 5 mL to each tissue collectioncontainer. The tissue collection containers were then placed into ashaking incubator at about 37° C., and about 60 rpm for about 30 min todisaggregate the unprocessed lipoaspirate. The disaggregated tissuesample was then passed through a 100 μm steriflip filter. The filteredtissue was then centrifuged at a rate of about 600×g for about 10minutes to recover regenerative cells. The recovered cells were placedin culture in minimum essential medium (MEM) with 20% (v/v) fetal bovineserum for 24 hours. The adherent cells were counted by a hemacytometer.

As illustrated in FIG. 10, extruding the tissue sample prior tocentrifugation yields a greater number of cells per gram of tissue. FIG.11 illustrates that centrifugation at 1200×g has an additive effect interms of increasing the cell concentration within the cell-enrichedmatrix regardless of whether the tissue was extruded prior tocentrifugation.

FIG. 12 illustrates that a longer amount of time for centrifugation ofthe tissue sample yields a higher number of cell per gram ofcell-enriched matrix. FIG. 13 illustrates that a 1200×g rate ofacceleration yields a higher concentration of cells per cell-enrichedmatrix when compared to a 400×g rate of acceleration or nocentrifugation at all.

Example 4

Fresh human lipoaspirate was divided into two aliquots. Aliquot 1(“control method”) was processed using conditions similar to thosecommonly employed in cosmetic surgery to prepare lipoaspirate derivedfat graft. The lipoaspirate was centrifuged for 2 minutes at 200×g.Aliquot 2 (“cell-enriched matrix” (CEM) method) was processed by firstextruding the lipoaspirate across a luer coupling between two syringes 5times, and then centrifuging the extruded lipoaspirate for 30 minutes at1200×g. After centrifugation, both methods resulted in fractionationinto an upper oil layer, a middle tissue layer, and a lower aqueouslayer. The middle tissue layer fraction of each aliquot was collected. Aportion of the collected tissue layer fraction from each method wasloaded into individual 1 cc syringes and administered subcutaneouslyinto the nuchal area of female immunodeficient NU/NU mice (n=3mice/preparation).

An additional portion of the tissue layer fraction from each method wasprocessed at 37° C. with a blend of collagenases I and II and dispase,filtered through a 100 μm filter, and then centrifuged at 600×g toobtain the regenerative cells. Number of viable regenerative cells inthe fresh cell preparations and number of plastic adherent cells inculture at 24 h were determined. At 1 month post-implantation, mice weresacrificed and grafts were evaluated.

Processing by the CEM method resulted in a 2.2 fold higher concentrationof viable cells in the fresh preparation and a 5.5 fold higherconcentration of plastic adherent cells compared to the control method.See Table 1. This cell enrichment translated to a higher viability ofthe graft at 1 month as evidenced by vascularization and absence of oilpockets in mice injected with the cell preparations obtained using theCEM method.

TABLE 1 Method Viable cells/g tissue Adherent cells/g tissue Control2.82 × 10⁵ 5.35 × 10⁴ CEM 6.16 × 10⁵ 2.94 × 10⁵

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1. A cellular composition comprising the combination of (i) acell-enriched matrix prepared from a sample of body tissue havingvasculature and regenerative cells adaptive to differentiate intofunctional equivalent of host tissue after transplantation therein, byextruding the body tissue sample through an orifice to reduce lipidcontent and increase regenerative cell content in the sample, followedby centrifugation of the extruded sample at a centrifugal force andduration sufficient to derive therefrom the cell-enriched matrix withregenerative platform therein; together with (ii) a regenerative cellpopulation recovered by subjecting such a derived cell-enriched matrixto repetitive cycles of rotational acceleration and deceleration of atleast one cycle thereof per minute in a temperature-controlledcentrifuge with enzymatic environment to dissociate and extractregenerative cells from the matrix.
 2. The composition of claim 1wherein the body tissue is selected from one of lipoaspirate, adiposetissue, and combinations thereof.
 3. The composition of claim 1 whereincentrifugation of the extruded sample is applied with a force in a rangefrom about 400 g to about 2000 g for a duration of about 5 minutes. 4.The composition of claim 1 wherein the orifice through which the sampleof body tissue is extruded has a diameter in a range from about 1 mm toabout 5 mm.
 5. The composition of claim 1 wherein the rotationalacceleration and deceleration is performed by securing such acell-derived matrix in a container to an inverted rotor of a centrifuge,and subjecting the secured container to a sequence of multiple rounds ofthe rotational acceleration and deceleration of at least one round perminute at a centrifugal force in a predetermined range for a durationsufficient for the dissociation and extraction of regenerative cells. 6.The composition of claim 5 wherein the multiple cell-enriched matrixcontainers are secured radially in spaced-apart orientation to the rotorfor retention thereof at one of (i) a fixed angle or (ii) an angle thatvaries with rotational force on the respective container, and the rotoris spun under rotational forces through the sequential rounds ofrotational acceleration and deceleration at a rate of said at least oneround per minute, to dissociate regenerative cells from the containedcell-enriched matrix as a combined result of conflicting centrifugal andgravitational forces thereon.
 7. The composition of claim 6 wherein saidangle, whether fixed or varying with rotational forces on the container,is positively acute between and including a horizontal plane through therotational axis of the rotor and a vertical orientation of said axis soas to create said conflicting centrifugal and gravitational forcesduring start-stop cycles resulting from said sequential rounds ofrotational acceleration and deceleration.
 8. The composition of claim 1wherein the enzymatic environment in said temperature controlledcentrifuge is provided by a proteolytic enzyme selected from acollagenase, a neutral protease, or a combination thereof.
 9. Thecomposition of claim 5 wherein the temperature within said temperaturecontrolled centrifuge is maintained in a range from about 26° C. toabout 42° C. during said multiple rounds of rotational acceleration anddeceleration.
 10. The composition of claim 5 wherein the sequentialrounds of rotational acceleration and deceleration are substantiallyuninterrupted during centrifugation.
 11. The composition of claim 5wherein the cellular composition is suitable to be banked as is or in aresorbable scaffold, for subsequent implantation into a subject.
 12. Thecomposition of claim 11 wherein the banking is enabled by firstsubjecting the cellular composition or scaffold to cryopreservation. 13.The composition of claim 1 wherein said cell-enriched matrix and saidregenerative cell population exhibits enhanced neovascularization forlong term survival as a transplanted volume-filling graft.
 14. Thecomposition of claim 1 prepared for implantation in the same subjectfrom which said sample was originally taken.
 15. The composition ofclaim 11 wherein the subject to receive the implantation is differentfrom the subject from which said sample was originally acquired.
 16. Thecomposition of claim 10 wherein the cellular composition possesses acapability to restore cellular function of non-functioning tissue of asubject when implanted at a target site of the non-functioning tissue.